The ability to isolate exosomes, circulating tumor cells (CTCs), and other circulating factors from biological fluids in a precise, biocompatible, and convenient manner is critical for many biomedical studies and applications. Here we summarize our recent progress on an “acoustic tweezers” technology that utilizes sound waves to manipulate exosomes, CTCs, and other tiny particles. For example, we developed an acoustic separation method to isolate exosomes directly from undiluted whole blood in a label-free and contact-free manner. This device consists of two modules. Micro-scale blood components are first removed by the cell-removal module, followed by extracellular vesicle subgroup separation in the exosome-isolation module. In the cell-removal module, we demonstrate the isolation of 110 nm particles from a mixture of micro- and nano-sized particles with a yield greater than 99%. In the exosome-isolation module, we isolate exosomes from an extracellular vesicle mixture with a purity of 98.4%. Integrating the two acoustofluidic modules onto a single chip, we isolated exosomes from whole blood with a blood cell removal rate of over 99.999%. The acoustic tweezers technology is capable of delivering high-precision, high-throughput, high-efficiency cell/particle/fluid manipulation in a simple, inexpensive, cell-phone-sized device. More importantly, the acoustic power intensity and frequency used in the acoustic tweezers technology are in a similar range as those used in ultrasonic imaging, which has proven to be extremely safe for health monitoring, even during various stages of pregnancy. As a result, the acoustic tweezers technology is extremely biocompatible; i.e., cells can maintain their natural states and highest integrity during the acoustic cell-manipulation process.